CN110283835A

CN110283835A - 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A of one kind and construction method

Info

Publication number: CN110283835A
Application number: CN201910551575.5A
Authority: CN
Inventors: 文兴建; 程安春; 汪铭书
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2019-09-27
Anticipated expiration: 2039-06-24
Also published as: CN110283835B

Abstract

The invention discloses 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A of one kind and construction methods, the mutated gene ISA-T1142A-C4334A sports A by T with 3 the 1142nd nucleotide of type duck hepatitis A virus velogen strain genome, so that the 164th amino acids of viral VP0 albumen be made to sport asparagine by the tyrosine of parent plant；4334th nucleotide sports A by C, so that the 71st amino acids of viral 2C albumen be made to sport isoleucine by the leucine of parent plant.3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A mutant strains are one plant of ideal vaccine candidate strains；It can also be used to 3 type duck hepatitis A virus cause to have a extensive future in the basic research such as weak molecule mechanism.

Description

3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A of one kind and construction method

Technical field

The invention belongs to gene engineering technology fields, and in particular to 3 type duck hepatitis A virus mutated gene ISA- of one kind T1142A-C4334A and construction method.

Background technique

Duck virus hepatitis (Duck viral hepatitis, DVH) is by duck hepatitis virus (Duck hepatitis Virus, DHV) one kind is acute, highly contagious disease caused by infection duckling.Current duck culturing area main in the world has The presence of this disease has the characteristics that interval outburst, endemicity, is to endanger one of principal disease of duck culturing industry.The disease is main The duckling within four week old is encroached on, has morbidity anxious, is propagated rapidly, the features such as course of disease is short and the death rate is high；Clinical manifestation For the dead preceding generation spasm of duckling, head is swung back to back, is in " opisthotonos ", and pathological change is mainly the visible liver enlargement hair of dissect Scorching and a large amount of haemorrhagic puncta.The disease is mainly by belonging to the duck hepatitis A virus (Duck of Picornaviridae fowl hepatovirus Hepatitis Avirus, DHAV) cause.There are three types of serotype, i.e. 1 type, 2 types and 3 types for DHAV tool.In recent years, China is popular DHAV be mainly 1 type duck hepatitis A virus (DHAV-1) and 3 type duck hepatitis A virus (DHAV-3).

Reverse genetics manipulation technology is a kind of Important Platform for carrying out viral molecular biology research, can be right in vitro Rna virus cdna group carries out the manual operations such as gene knockout, direct mutagenesis, the energy in illustrating viral pathogenesis mechanism and vaccine development It enough plays a significant role and has the advantage shorter than the natural mutagenesis period.The key of traditional picornavirus infection cloning process is to obtain Full-length cDNA full length fragment clone, and viral genome be converted to cDNA after need to be cloned into suitable carrier In, in order to avoid instability problem of the virus sequence in bacterium, researcher is usually taken cDNA clones method, passes through small fragment Large fragment is connected into, large fragment is finally obtained into full length cDNA clone by the method that digestion connects.But this method digestion position It is more that point chooses limitation, and multiple large fragments are attached to efficiency is lower, and other rdrp gene cDNA clone exists in vitro There is unstability, therefore not only operating procedure is troublesome for the whole process of acquisition full length viral genome cDNA in bacterium, but also It taking a long time, success rate is not high, meanwhile, although the full-length cDNA of some viruses can not be cloned or can be cloned into carrier in place It easily morphs in main bacterium and leads to the Revive virus that cannot succeed.Currently, a kind of be referred to as " infectious Subgenomic replicon The technology of (Infectious Subgenomic Amplicons) " has been shown to real in mammal or mosquito cells Now to the artificial rescue of single strand plus RNA virus, and it is applied to japanese encephalitis virus, west nile virus, zika virus, yellow heat In the reverse genetics research of the virus such as virus, dengue virus and people's coxsackie virus.The technology is a kind of novel " without thin Bacterium " reverse-genetics approach does not need to obtain virus full length cDNA plasmid and obtains transcription of viral RNA sheet in vitro, can be with Directly there is by transfection the DNA fragmentation Revive virus of homology region.For specific, " infectious sub-genome duplication The technology path that son " uses is: generating the non-infectious subunit comprising entire virus genomic overlapping by PCR method first Because of a group DNA fragmentation, the Subgenomic replicon quantity of these overlappings can be 3 to 10, have between adjacent replicon The overlapping region of 100bp or so, meanwhile, the 5' of first segment and the end 3' of the last one segment flank giant cell disease respectively Malicious early promoter (Cytomegalovirus immediate early promoter, pCMV) sequence, Hepatitis D virus Ribozyme (Hepatitis delta virus ribozyme, HDVR) sequence and vacuolating virus of monkey early stage mRNA polyadenylation letter Number (SV40 early mRNA polyadenylation signal, SV40pA) sequence, these elements can help sub-gene Group replicon be mixed be transfected into permissive cell after start to transcribe, and using host cell homologous recombination machinery it is spontaneous heavy Group, being formed has infective intact virus transcript, then leads to the duplication and proliferation of virus, and final obtain has infectivity Revive virus.

DHAV-1 attenuated vaccine mainly is used to the prevention and control of DHAV on Vehicles Collected from Market, still lacks efficient DHAV-3 epidemic disease living Seedling is researched and developed and is suitble to the New molecular marker vaccine of China DHAV-3 popularity extremely urgent；Meanwhile DHAV host's preferendum and poison The viral gene of power variation and the basic research such as critical sites can provide theoretical foundation for the prevention and treatment of duck hepatitis, this also there is an urgent need to Obtain the Strain of host's preferendum and virulence variation.

Summary of the invention

The technical problem to be solved by the present invention is to obtain 3 type duck hepatitis A virus epidemic disease of molecular labeling using genetic modification platform Seedling candidate's strain；Meanwhile the Strain for obtaining virulence variation can be the viral gene and critical sites of research DHAV virulence variation Deng offer basic material.

In order to achieve the above technical purposes, the present invention is realized especially by following technical scheme:

3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A, the mutated gene ISA-T1142A- of one kind C4334A sports A by T with the 1142nd nucleotide of 3 type duck hepatitis A virus velogen strain genomes, to make viral VP0 egg The 164th white amino acids sport asparagine by the tyrosine of parent plant；4334th nucleotide sports A by C, thus The 71st amino acids of viral 2C albumen are made to sport isoleucine by the leucine of parent plant.

The 3 type duck hepatitis A virus (Duck Hepatitis A Virus type 3, DHAV-3) are preserved in China Type Tissue Collection, deposit number are CCTCC NO:V201305, preservation address: Wuhan City, Hubei Province Wuchang District Aug. 1st The Wuhan University of road 299 in the school, preservation date: on March 25th, 2013.

The G of the 3403rd nucleotide of mutated gene genome sports T, the heredity mark as infection clones Note, can distinguish mutated gene and parent plant and street strain by PCR method combination DNA sequencing.

The mutated gene Strain that the present invention contains genetic marker can be steady in 9 age in days duck embryos as parental virus Fixed proliferation passage, virus titer is higher, and genetic stability is good, is not mutated for continuous passage 10 times.

The present invention contains the mutated gene Strain of genetic marker relative to parental virus, and mutated gene is multiple in duckling body It makes but not pathogenic to duckling, there is good safety, can be used as 3 type duck hepatitis A virus vaccine candidate strains.

In another aspect of this invention, it provides above-mentioned mutated gene and is preparing the application in 3 type duck hepatitis virus vaccines.

The mutated gene can be also used in duck hepatitis virus virulence and cause in weak Study on Molecular Mechanism simultaneously.

In another aspect of this invention, the construction method of above-mentioned mutated gene mutant strain is provided, comprising the following steps:

(1) parental virus full-length genome is divided into (2.6kb, 2.6kb and 2.7kb) three segments similar in size to carry out PCR amplification adds cytomegalovirus early promoter (Cytomegalovirus at the end 5' of first segment of viral genome Immediate early promoter, pCMV) and introduce in VP0 gene mutational site, in the 2C gene of second segment It introduces mutational site and the nonsense mutation of 2A gene is as genetic marker site, added at the end 3' of viral genome the third fragment Hepatitis delta virus ribozyme (Hepatitis delta virus ribozyme, HDVR) sequence and vacuolating virus of monkey early stage MRNA polyadenylation signal (SV40 early mRNA polyadenylation signal, SV40pA) sequence obtains three A DNA fragmentation constitutes 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A " infectious Subgenomic replicon "；

(2) by 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A " infectious sub-genome duplications of building Son " transfects duck embryo fibroblasts after mixing with transfection reagent, replicon transcribes in host cell and utilizes the homologous of cell The spontaneous recombination of recombination mechanism, being formed has infective intact virus transcript, then leads to the duplication and proliferation of virus, finally Obtain the 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A mutant strains containing genetic marker.

Further, first segment and the third fragment contain 74 and 83 base-pairs with second segment respectively Overlapping region.

The invention has the benefit that

1,3 type duck hepatitis A virus mutated genes are obtained than natural method mutagenesis and conventional counter heredity using the method for the present invention The time cycle of operating technology is short, can accelerate the cultivation and viral pathogenesis Mechanism Study of 3 type duck hepatitis A virus attenuated vaccine strains Process.

2, in the present invention low virulent strain ISA-T1142A-C4334A that obtains have antigenicity similar with its parent's strain and Stable hereditary capacity is able to maintain during continuous passage, therefore the mutated gene mutant strain can be used as 3 type duck hepatitis As Malicious vaccine candidate vaccine strain；It has proliferation efficiency more higher than parent plant and virus titer in duck embryos simultaneously, is such as used for In production of vaccine, yield can be improved, reduce cost.

3, the duplication but not pathogenic to duckling in duckling body, therefore the mutated gene mutant strain can be used as 3 type duck hepatitis As Malicious attenuated vaccine candidate strain has good safety.

4, since the mutated gene ISA-T1142A-C4334A mutant strain obtained in the present invention is compared with parent plant, virulence In the application of the basic research such as viral gene and the critical sites for reducing, therefore can be used for the variation of duck hepatitis virus virulence.

Detailed description of the invention

Fig. 1 be based on 3 type duck hepatitis A virus be framework construction molecular labeling mutated gene ISA-T1142A-C4334A " sense The schematic diagram of metachromia Subgenomic replicon "；

Fig. 2 is 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A " infectious Subgenomic replicon " transfection Duck embryo fibroblasts result figure；

Fig. 3 is mutated gene ISA-T1142A-C4334A virus genomic 3403rd nucleic acid molecule genetic marker position Point sequencing result；

Fig. 4 is mutated gene ISA-T1142A-C4334A virus genomic 1142nd nucleotide targeted mutagenesis site core Thuja acid sequencing result；

Fig. 5 is mutated gene ISA-T1142A-C4334A virus genomic 4334th nucleotide targeted mutagenesis site core Thuja acid sequencing result.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples unless otherwise specified can be from Commercial sources obtain.

Material and reagent as used in the following examples are specific as follows:

Strain:

3 type duck hepatitis A virus velogen strains: this laboratory is isolated, has been preserved in the Chinese Typical Representative training of Wuhan, China university Support object collection, deposit number are as follows: CCTCC NO:V201305, classification naming: 3 type duck of fowl hepatovirus Picornaviridae Hepatitis A virus (Duck Hepatitis AVirus type 3, DHAV-3).

Reagent and instrument:

TaKaRa MiniBEST Universal RNA Extraction Kit, PrimeSTAR Max DNA Polymerase, DNA Marker etc. is purchased from precious bioengineering (Dalian) Co., Ltd；Plastic recovery kit, plasmid extraction reagent Box etc. is purchased from U.S. Omega company；Lipofectamine box Lipofectamine 3000 is purchased from Invitrogen company；Its His reagent is that domestic analysis is pure.

Nucleic acid-protein detector (Bio Rad, Smartspec 3000), grads PCR instrument (Biometra, Tgradient), electrophoresis apparatus (Bio Rad, Powerpac 300) and gel imaging system (Bio Rad Versa Doc Model 2000)。

13 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A's " infectious Subgenomic replicon " of embodiment Building and virus rescue

1.1. the design and synthesis of primer

According to the whole genome sequence of 3 type duck hepatitis A virus in GenBank, it is viral complete for expanding to devise 8 pairs of primers Genome sequence, pCMV and SV40pA sequence, particular sequence information are shown in Table 1, and primer is by Shanghai Sheng Gong bioengineering Co., Ltd Synthesis.

Table 1 constructs 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A infectivity Subgenomic replicon primers

1.2. virus extracting

It is operated referring to the operation instructions of TaKaRa MiniBEST Universal RNA Extraction kit, from The viral full-length genome RNA of 3 type duck hepatitis A virus separation strains is extracted in duck embryos allantoic fluid, and uses nucleic acid-protein detector After (Bio Rad, Smartspec3000) measures its nucleic acid concentration and purity, -70 DEG C are saved backup.

1.3. gene fragment amplification is cloned

(1) using PrimeScript II 1st Strand cDNA Synthesis kit that the total serum IgE of extracting is anti- It is transcribed into cDNA template, DNA High fidelity PCR enzyme PrimeSTAR Max DNA Polymerase is then used, uses primers F 1- F and T1142A-R obtains DHAV-3-F1-T1142A-R piece by template amplification of the reverse transcription product of parent plant virus total RNA Section, obtains DHAV-3-F1- as template amplification using the reverse transcription product of parent plant virus total RNA using T1142A-F and F1-R T1142A-F segment, using primers F 2-1-F and F2-1-R, F2-2-F and C4334A-R, C4334A-F and F2-2-R with parent The reverse transcription product of strain virus total serum IgE expands respectively for template and obtains DHAV-3-F2-1 segment, DHAV-3-F2-C4334A-1 piece Section and DHAV-3-F2-C4334A-2 segment, using primers F 3-HDVR-F and F3-HDVR-R with the anti-of parent plant virus total RNA Transcription product is that template amplification obtains DHAV-3-F3-HDVR segment；Using eukaryon expression plasmid pEGFP-C1 as template, using drawing Object pCMV-F and pCMV-R expand to obtain pCMV segment, expand to obtain using primer HDVR-SV40pA-F and HDVR-SV40pA-R HDVR-SV40pA segment.

(2) by fusion DNA vaccine technology, as shown in Figure 1, first by DHAV-3-F1-T1142A-F and DHAV-3-F1- T1142A-R segment composition is DHAV-3-F1-T1142A, is then by pCMV and DHAV-3-F1-T1142A segment composition pCMV-F1；By DHAV-3-F2-1 segment, DHAV-3-F2-C4334A-1 segment and DHAV-3-F2-C4334A-2 segment composition For F2 segment；It is F3-HdvRz/SV40pA segment by DHAV-3-F3-HDVR and HDVR-SV40pA segment composition, shown in Fig. 1, Three DNA fragmentations finally obtained constitute mutated gene ISA-T1142A-C4334A " infectious Subgenomic replicon ".Expand Increase segment after the separation of 1% agarose gel electrophoresis, gel reclaims kit (Omega) gel extraction purpose piece is respectively adopted Section.DNA fragmentation is sent to Shanghai Sheng Gong bioengineering Co., Ltd and is sequenced.

1.4. the transfection rescue of mutated gene ISA-T1142A-C4334A " infectious Subgenomic replicon "

Primary duck embryo fibroblasts are prepared using 9 age in days duck embryos, when the cell in 3.5cm culture dish is grown to 90% fusion when, by pCMV-F1, F2 and F3-HdvRz-SV40pA genetic fragment of equivalent (1.5 μ g) with 90% duck embryo fibroblasts are covered in transfection after Lipofectamine3000 (Invitrogen) mixing, and control group is used only Lipofectamine 3000 (Invitrogen) is transfected.Cell is placed in 37 DEG C of 5%CO₂Culture sight is carried out in incubator It examines, replaces culture medium after 16 hours, after transfection 72 hours, cell fragmentation phenomenon occurs in transfection group cell, and cellular control unit Upgrowth situation is good.After transfection duck embryo fibroblasts 120 hours, after cell growth condition is as shown in Fig. 2, photograph to record By cell multigelation 3 times, cell culture fluid is inoculated with 5 piece of 9 age in days duck embryos by allantoic cavity approach, and 0.2mL/ pieces, paraffin sealing After be put into and continue to be incubated in incubator, every 8 hours photograph embryo 1 time, the death condition of observation duck embryos after inoculation is discarded 24 hours Interior death duck embryos.Duck embryos are dead between 24 hours to 72 hours after inoculation as the result is shown, and duck embryos death idiosome bleeding is serious, Allantoic fluid is collected to be saved as first generation reverse genetic Strain.

The identification and characteristic of 23 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A of embodiment

2.1. in Revive virus genetic marker identification

It may be from parental virus or the possibility of street strain's pollution in transfection or succeeding generations to exclude Revive virus, Using reverse-genetics approach, 3403 bit bases of mutated gene genome sport T by being mutated G, which does not change 2A egg The composition of Bai Xiangying amino acid, the site can be distinguished as molecular genetic marker site by PCR method combination DNA sequencing Mutated gene mutant strain and parent plant and street strain.Revive virus passes through limiting dilution assay passage purifying 5 times in duck embryos.From Total serum IgE is extracted in allantoic fluid, expands the DNA piece containing genetic marker site using F2-F and F2-R primer PCR after reverse transcription Section, amplified fragments are separated through 1% agarose gel electrophoresis, then use gel reclaims kit (Omega) gel extraction, and will DNA fragmentation send to Shanghai Sheng Gong bioengineering Co., Ltd and is sequenced, and the product of sequencing result display amplification contains introducing Silent mutation (G3403T), as shown in figure 3, show we obtain correct Revive virus, rather than parent plant or wild type Strain pollution.

2.2. the genetic stability in Revive virus and its mutational site detects

For observe save out reverse genetic virus whether passage can be proliferated in duck embryos, by the rescue of 1st generation Virus does 1:100 dilution with sterile saline, is inoculated with 5 piece of 9 age in days duck embryos.The duck embryos death time concentrates on being inoculated with as the result is shown Afterwards between 24 hours to 72 hours, idiosome lesion is obvious, and continuous passage 10 times, during which collects every generation virus liquid, is put in -80 DEG C refrigerator saves.RNA is extracted using the generation virus liquid of the 1st, 5 and 10, after reverse transcription is cDNA, PCR amplification contains genetic marker position The DNA fragmentation of point, and genetic marker site is detected, as shown in Figure 4 and Figure 5, sequencing result shows the generation of the 1st, 5 and 10 poison It is not mutated, shows that this viral genetic has good stability.

2.3. virus multiplication and assay

With 3 type duck hepatitis A virus Revive virus of sterile saline solution doubling dilution and parent plant, 10 are selected^-3、10^-4、 10^-5、10^-6、10^-7、10^-8This 6 dilutions are inoculated with the duck of 5 piece of 9 age in days with the amount of every embryo 0.2mL through allantoic cavity approach respectively Embryo separately sets sterile saline and compares 5 pieces, and inoculation is placed in 37 DEG C of constant incubators and is incubated for, and dead duck embryos are not in 24 hours Meter, observes and records the death and survival condition that duck embryos are inoculated in 7 days, calculates ELD50 by Reed-Muech method, as the result is shown Revive virus has different proliferative capacities from parent's strain virus, and viral level is respectively 10 in every 0.2mL allantoic fluid^-8.37ELD50 With 10^-4.50ELD50 shows that the proliferative capacity of 3 type duck hepatitis A virus mutated gene mutant strains is significantly stronger than parent plant, is such as used for Antigen production can increase yield, reduce cost in vaccine.

2.4. the antigenicity of serum neutralization test detection virus

Mature progeny viral particles and virus antigenicity whether are generated in duck embryos passage for detection reverse genetic strain Whether change.Using fixed virus diluted blood heat-clearing method measurement 3 type duck hepatitis A virus serum of rabbit-anti to mutated gene mutant strain With the neutralization titer of parent plant, the 3 type duck hepatitis A virus standard serum of rabbit-anti for first preparing laboratory early period where inventor (potency >=1:128) makees 10 times of doubling dilutions from 2 with sterile saline^-1To 2^-9This 9 dilutions, while by viral dilution Both then contain 200ELD50 at each unit dose (0.2mL), mixed in equal amounts and be added dual anti-37 DEG C of penicillin and streptomycin of 5% It water-bath 1 hour, is then inoculated in 9 age in days health duck embryos allantoic cavities with the metering of every embryo 0.2mL, each dilution is inoculated with 5 pieces of ducks Embryo.Separately setting negative serum control group (healthy rabbit anteserum with virus mix) and blank control group, (sterile saline is mixed with virus Close), the case where discarding duck embryos dead in 24 hours, observe and record the duck embryos death and survival in 7 days, then calculate rabbit Neutralization titer of the anti-3 type duck hepatitis A virus standard serums to virus.

The result shows that Revive virus and parent's strain virus antigenicity having the same, negative serum control group (Healthy Rabbits blood Mix with virus clearly) and the duck embryos of blank control group (sterile saline with viral mix) it is complete to 48 small times at 24 hours Portion is dead；When serum dilution is 2^-1To 2^-6Between when, in mutated gene mutant strain and parent plant and the duck embryos of group are all strong It is living, when dilution is 2^-7When duck embryos start to lose protection, start at this time, higher with dilution, duck embryos protective rate is lower, until dilute Degree of releasing is 2^-9When duck embryos thoroughly lose protection.Show that 3 type duck first can also be protected by such as preparing vaccine antigen with mutated gene mutant strain Hepatovirus infection.

2.5. to the virulence and safety testing of susceptible duckling

Safety testing is carried out using parent plant and mutated gene mutant strain, acquires the liver of dead duck embryos in embodiment 2.3 Dirty tissue homogenized, sterile phosphate buffer solution is added in the ratio of 1:100 by volume, grinds, multigelation 3 times, 12000g is centrifuged 10min, and supernatant is inoculated with 9 age in days duck embryos through allantoic cavity approach after 0.22 μm of filter filtration sterilization and measures it Then virus liquid is diluted to 10 by ELD50^3.0ELD50/0.4mL.In addition 1 age in days health duckling 30 is only randomly divided into 3 groups, examination The duckling for testing group is inoculated with 0.4mL parent plant or mutated gene mutant strain, every inoculation 10 through intramuscular injection path^3.0ELD50/ 0.4mL, the duckling of control group are then inoculated with isometric sterile saline, and the isolated rearing in different animals room, free water is adopted Food, is observed daily after inoculation, records morbidity, the death condition of duckling, timely dissect death duckling, dissect is survived after observation 7 days Duckling, the lesion situation of record duckling liver, kidney and other organs.

7 days internal reference group ducklings do not occur clinical symptoms, and duckling feed and drinking-water situation are normal, duckling after parent plant inoculation Disease incidence 80%, the death rate 60%, dead duck dissect observe that typical duck hepatitis disease is presented in duckling liver, kidney and other organs Become；And the duckling for being inoculated with mutated gene mutant strain does not occur clinical symptoms, feed and drinking-water situation are normal, and in cloacal swab Detect virus, show that mutated gene mutant strain has been decreased obviously duckling pathogenicity, in duckling body duplication but to duckling not It causes a disease, has using it as the potentiality of duck hepatitis virus attenuated vaccine strain, and can be distinguished by PCR method combination DNA sequencing Mutated gene mutant strain and parent plant.And the virulence of the mutated gene mutant strain is changed, and can be used as duck hepatitis virus The basic material of the researchs such as the viral gene and its critical sites of virulence variation.

33 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A mutant strain of embodiment is in preparing inactivated vaccine Using

There is higher proliferation efficiency than parent plant in duck embryos according to the mutated gene mutant strain measured in embodiment 2 And virus titer；Immunogenicity and genetic stability are good；Duckling pathogenicity has been decreased obviously, has shown mutated gene ISA- T1142A-C4334A mutant strain can be used as a strain vaccine Candidate Strain and be used to prepare 3 type duck hepatitis A virus vaccines.

The preparation method of 3.1 inactivated vaccines

Using 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A mutant strains as kind of a poison sterile saline 100 times of dilution, allantoic cavity are inoculated with 20 piece of 9 age in days duck embryos, and every 0.2 mL of embryo sets in 37 DEG C of constant incubators and continues to be incubated for, and are inoculated with It is shone egg 1 time after 24 hours afterwards, discards dead germ, later every 8 hours photograph egg 1 time, dead duck embryos taken out at any time, until 48 is small When, duck embryos are all dead, and the duck embryos gas chamber of collection is upright upwards, 4 DEG C of coolings 8 hours are placed in, by sterile collection after cooling Duck embryos allantoic fluid is placed in -20 DEG C and saves backup.

Virus liquid is handled using final concentration of 0.1% formalin, 37 DEG C inactivate 24 hours, then use sterile physiological Viral level is diluted to 10 by salt water³It emulsifies mixing after ELD50/0.1mL in equal volume with incomplete Freund's adjuvant, that is, 3 types is made Duck hepatitis A virus mutated gene mutant strain inactivated vaccine.

3.2 sterile and detection of mycoplasma

Inactivated vaccine is carried out sterile and mycoplasma according to existing " Republic of China Veterinary Pharmacopoeia " annex to examine, detection Result is feminine gender.

The detection of 3.3 exogenous virus

Inactivated vaccine is subjected to exogenous virus detection, testing result according to existing " Republic of China Veterinary Pharmacopoeia " annex It is feminine gender.

The safety detection of 3.4 inactivated vaccines

For the safety for examining inactivated vaccine, 10 1 age in days ducklings are immunized with 10 times of immunizing doses, every through leg muscle Route of inoculation is inoculated with 0.2mL, while taking 10 duckling injection sterile salines as a control group, isolated rearing, free water Feeding observes and records the health condition of duckling daily, and observation analysed duckling after 7 days, and as a result immune group is connecing with control group duckling Without morbidity in kind 7 days, and analyse the results show that not occurring pathology change in each immune organ of duckling and virus tropism tissue Change, and virus is not detected in cloacal swab, shows that inactivated vaccine is not pathogenic to 1 age in days duckling.

The immune efficacy of 3.5 inactivated vaccines

1 age in days duckling 20 is only randomly divided into 2 groups, and 10 ducklings of inactivated vaccine inoculation group are through leg muscle injecting pathway It is inoculated with the above-mentioned vaccine immunity being prepared of a plumage part, another group of injection equivalent sterile saline is as control, two groups of ducklings The case where isolated rearing, free water searches for food, observes and records duckling after inoculation daily, with 10 times of LD50 dosage parent plants after 7 days It carries out attacking poison, attacks after poison and observe and record morbidity, the death condition of duckling daily, timely dissect death duckling, continuous observation 1 week, Dissect survival duckling, the lesion situation of record liver, kidney and other organs.

Experimental group and negative control group duckling do not occur clinical symptoms, duckling feed and drinking-water situation in 7 days after immune Normally.The 2nd day negative control group has duckling lassitude after attacking poison, nervous symptoms occurs, until the 7th day, totally 6 death, survival Duckling has the lesions such as different degrees of liver bleeding, disease incidence 80% (8/10), the death rate 60% (6/10), and inactivated vaccine is exempted from Epidemic disease group duckling is dead without morbidity, and normal water feeding shows that the inactivated vaccine prepared with mutated gene mutant strain is safe and effective, can Homologous virulent poison is attacked to protect.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, although referring to aforementioned reality Applying example, invention is explained in detail, for those skilled in the art, still can be to aforementioned each implementation Technical solution documented by example is modified or equivalent replacement of some of the technical features.It is all in essence of the invention Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Sequence table

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<210> 17

<211> 583

<212> DNA

<213>cytomegalovirus early promoter is sub (pCMV)

<400> 17

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540

acggtgggag gtctatataa gcagagctgg tttagtgaac cgt 583

<210> 18

<211> 67

<212> DNA

<213>hepatitis delta virus ribozyme sequence (HDVR)

<400> 18

gggtcggcat ggcatctcca cctcctcgcg gtccgacctg ggctacttcg gtaggctaag 60

ggagaag 67

<210> 19

<211> 122

<212> DNA

<213>vacuolating virus of monkey early stage mRNA polyadenylation signal (SV40pA)

<400> 19

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120

ta 122

Claims

1. 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A of one kind, which is characterized in that the mutated gene ISA-T1142A-C4334A sports A by T with the 1142nd nucleotide of 3 type duck hepatitis A virus velogen strain genomes, to make 164th amino acids of viral VP0 albumen sport asparagine by the tyrosine of parent plant；4334th nucleotide is dashed forward by C Become A, so that the 71st amino acids of viral 2C albumen be made to sport isoleucine by the leucine of parent plant.

2. a kind of 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A according to claim 1, feature exist In the 3 type duck hepatitis A virus velogen strains are preserved in China typical culture collection center, and deposit number is CCTCC NO: V201305。

3. a kind of 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A according to claim 1, feature exist In the G of the 3403rd nucleotide of mutated gene genome sports T, the genetic marker as infection clones.

4. the construction method of 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A mutant strain described in claim 1, Characterized by comprising the following steps:

1) parental virus full-length genome is divided into three segments similar in size and carries out PCR amplification, at viral genome first The end the 5' addition cytomegalovirus early promoter of segment is sub and mutational site, the 2C base of second segment are introduced in VP0 gene Mutational site is introduced because in, adds hepatitis delta virus ribozyme sequence and monkey vacuole at the end 3' of viral genome the third fragment Viral early stage mRNA polyadenylation signal sequence obtains three DNA fragmentations and constitutes 3 type duck hepatitis A virus mutated gene ISA- T1142A-C4334A infectivity Subgenomic replicon；

2) by 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A infectivity Subgenomic replicons of building with turn Duck embryo fibroblasts are transfected after transfection reagent mixing, replicon transcribes in host cell and utilizes the homologous recombination machinery of cell Spontaneous recombination, being formed has infective intact virus transcript, then leads to the duplication and proliferation of virus, is finally contained 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A mutant strains of genetic marker.

5. construction method according to claim 4, which is characterized in that first segment and the third fragment are respectively with second A segment contains the overlapping region of 74 and 83 base-pairs.

6. construction method according to claim 4, which is characterized in that it is prominent to introduce nonsense in the 2A gene of second segment It is changed into as genetic marker site.

7. 3 type duck hepatitis A virus mutated gene ISA-T1142A-C4334A described in claim 1 is in 3 type duck hepatitis disease of preparation Application in malicious vaccine.